Modified biological control agents and their uses

ABSTRACT

Methods for improving the ability of a population of biological agents to compete and survive in a field setting are provided. By improving the population of biological agents, the modified population of agents is able to grow, compete with other microbial strains and fungi, and provide protection for plants from pathogens. In particular, modified biological agents and modified populations of such agents that are herbicide tolerant or resistant are selected or engineered. In this manner, the protection from disease-causing agents is enhanced. Such modified populations of biological agents can be added to soils to prevent fungal pathogens and the diseases they cause promoting plant growth. Therefore, the present invention is useful for enhancing the competitiveness of modified biological agents particularly over other microbial agents which are not herbicide resistant. Compositions of the invention include selected or engineered herbicide resistant biological agents and modified populations of biocontrol agents. These modified biological agents can be used as an inoculant or as a seed coating for plants and seeds.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application Ser.No. 61/933,954, filed Jan. 31, 2014 and U.S. Provisional ApplicationSer. No. 62/104,122, filed Jan. 16, 2015, the contents of bothapplications are herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to modified biocontrol agents and populations thathave improved properties.

BACKGROUND

Plant diseases and pests need to be controlled to maintain the qualityand quantity of food, feed, and fiber produced by growers around theworld. Plant diseases are mainly caused by fungi, bacteria, viruses andnematodes. Plant pests include chewing, sucking and piercing insectsfrom the Lepdoptera, Coleoptera, and Hemiptera, among others. Chemicalpesticides are widely used in farming to protect crops from such pestsand diseases. These chemical products fight crop pests, diseases, andweeds, resulting in improved yield. Without crop protection and pestcontrol, food production and the quality of food produced would decline.However, the use of chemical pesticides does impose a level of risk asmany have properties that can endanger health and the environment if notused properly.

A problem with the continued use of pesticides, herbicides, or othercrop protection chemicals is the development of resistance to thecontrol agent. Pesticide resistance is the decreased susceptibility of apest population to a control agent at doses that once killed mostindividuals of the species. Therefore, new products are needed withdifferent modes of action to aid in resistance management.

It has long been known that phylogenetically diverse microorganisms canact as natural antagonists of various plant pathogens and pests.Interactions between plant hosts and microorganisms that lead tobiocontrol can include antibiosis, competition, induction of hostresistance, and predation. Screening and testing isolates have yielded anumber of candidates for commercialization. Microbial biopesticidesrepresent an important option for the management of plant diseases andpests. There is a need for biological control agents that are able tocompete in field conditions particularly in the presence of herbicidesand fungicides that are commonly used in commercial farming and can haveantibiotic effects on microorganisms.

SUMMARY

Compositions and methods for improving the ability of a population ofbiological agents or biocontrol agents to compete and survive in a fieldsetting are provided. By improving the population of biological agents,the modified population of agents is able to grow, compete with othermicrobial strains and fungi, and provide protection for plants frompathogens. In addition, modified biological control agents promote plantgrowth and yield. In particular, modified biological agents and modifiedpopulations of such agents that are biocide-tolerant or -resistant;herbicide-tolerant or -resistant; fungicide-tolerant or -resistant;pesticide-tolerant or -resistant; or tolerant or resistant to cropprotection chemicals are selected or engineered. In this manner, theprotection of crops from disease-causing agents or pests is enhanced.

The modified biological agents are able to grow in the presence at leastone herbicide, fungicide, pesticide, or other crop protection chemicalthat is used in commercial farming. Such modified biological agents areable to grow and reproduce in soils where such herbicides, fungicides,pesticides, or other crop protection chemicals have been applied. Themodified biological agents render the soils suppressive or resistant todisease-causing pathogens or pests. Such modified populations ofbiological agents can be added to soils to prevent fungal pathogens andthe diseases they cause, or to inhibit feeding by insect pests ornematodes, promoting plant growth and increasing crop yield. Therefore,the present invention is useful for enhancing the competitiveness ofmodified biological agents particularly over other microbial agentswhich are not resistant to herbicides, fungicides, pesticides, or othercrop protection chemicals. Therefore, compositions of the inventioninclude selected or engineered biological agents and modifiedpopulations of biocontrol agents. These modified biological agents canbe used as an inoculant or as a seed coating for plants and seeds. Theycan also be applied as a spray application directly to the aerial partsof plants, and can be mixed with the herbicide or other chemical towhich they have been modified to become tolerant. As indicated, thepresence of the modified biological agents under field conditionsenhances resistance of the plants to pathogens and promotes plantgrowth. Such modified biological agents of the invention can be usedwith other agents to promote plant growth and yield.

Embodiments of the invention include:

1. A method for improving a biocontrol agent, said method comprisingmodifying said biocontrol agent to be resistant to at least oneherbicide, fungicide, pesticide, or other crop protection chemical.

2. The method of embodiment 1, wherein said biocontrol agent is modifiedby growing in the presence of a herbicide, fungicide, pesticide, orother crop protection chemical to select a resistant strain.

3. The method of embodiment 1, wherein said biocontrol agent is modifiedby transforming said biocontrol agent with a gene that confersresistance to said herbicide, fungicide, pesticide, or other cropprotection chemical.

4. The method of any one of embodiments 1-3, wherein said biocontrolagent is a bacterial biocontrol agent.

5. The method of any one of embodiments 1-3, wherein said biocontrolagent is selected from the group consisting of Pseudomonas, Bacillus,Agrobacterium, Lysobacter, Trichoderma, Paecilomyces, Gliocladium,Ampelomyces, Pythium, Metschnikowia, Chromobacterium, Penicillium,Coniothyrium, Chaetomium, Myrothecium, Aureobasidium, Pantoea,Burkholderia, Streptomyces, Variovorax, Pasteuria, Lactobacillus,Paenibacillus, Xanthomonas genera.

6. The method of embodiment 5, wherein said bacterial biocontrol agentis a Pseudomonas bacterium.

7. The method of embodiment 6, wherein said Pseudomonas is Pseudomonasfluorescens or Pseudomonas chlororaphis.

8. The method of any one of embodiments 1-7, wherein said herbicide isselected from the group consisting of glyphosate, glufosinate (glutaminesynthase inhibitor), sulfonylurea and imidazolinone herbicides (branchedchain amino acid synthesis inhibitors).

9. A modified biocontrol agent wherein said biocontrol agent has beenselected under herbicide, fungicide, pesticide, or other crop protectionchemical pressure and is resistant to said herbicide, fungicide,pesticide, or other crop protection chemical.

10. The modified biocontrol agent of embodiment 9, wherein said modifiedbiocontrol agent is a bacterial biocontrol agent.

11. The modified biocontrol agent of embodiment 9, wherein saidbiocontrol agent is selected from the group consisting of Pseudomonas,Bacillus, Agrobacterium, Lysobacter, Trichoderma, Paecilomyces,Gliocladium, Ampelomyces, Pythium, Metschnikowia, Chromobacterium,Penicillium, Coniothyrium, Chaetomium, Myrothecium, Aureobasidium,Pantoea, Burkholderia, Streptomyces, Variovorax, Pasteuria, Xanthomonasgenera.

12. The modified biocontrol agent of embodiment 10, wherein saidbacterial biocontrol agent is a Pseudomonas bacterium.

13. The modified biocontrol agent of embodiment 12, wherein saidPseudomonas is Pseudomonas fluorescens or Pseudomonas chlororaphis.

14. The modified biocontrol agent of any one of embodiments 9-13,wherein said herbicide is selected from the group consisting ofglyphosate, glufosinate (glutamine synthase inhibitor), sulfonylurea andimidazolinone herbicides (branched chain aminio acid synthesisinhibitors).

15. A recombinant biocontrol agent wherein said biocontrol agent hasbeen transformed with a herbicide resistance gene rendering thebiocontrol agent herbicide resistant.

16. The recombinant biocontrol agent of embodiment 15, wherein saidmodified biocontrol agent is a bacterial biocontrol agent.

17. The recombinant biocontrol agent of embodiment 16, wherein saidbacterial biocontrol agent is selected from the group consisting ofPseudomonas, Bacillus, Agrobacterium, Lysobacter, Gliocladium, Pythium,Chromobacterium, Penicillium, Pantoea, Burkholderia, Streptomyces,Variovorax, Pasteuria, and Xanthomonas genera.

18. The recombinant biocontrol agent of embodiment 17, wherein saidbacterial biocontrol agent is a Pseudomonas bacterium.

19. The recombinant biocontrol agent of embodiment 18, wherein saidPseudomonas is Pseudomonas fluorescens or Pseudomonas chlororaphis.

20. The recombinant biocontrol agent of any one of embodiments 15-19,wherein said herbicide is selected from the group consisting ofglyphosate, glufosinate (glutamine synthase inhibitor), sulfonylurea andimidazolinone herbicides (branched chain aminio acid synthesisinhibitors).

21. A modified population of biocontrol agents wherein the populationsubstantially comprises the biocontrol agent of any one of embodiments1-20.

22. A formulation for controlling a plant pathogen, said formulationcomprising a modified population of biocontrol agents, wherein saidbiocontrol agents are herbicide resistant, and a suitable carrier.

23. The formulation of embodiment 22, wherein said population comprisesmodified bacterial biocontrol agents.

24. The formulation of embodiment 22, wherein said population comprisesrecombinant biocontrol agents.

25. The formulation of any one of embodiments 22-24, wherein saidbiocontrol agent is present in an effective amount sufficient to improveplant health, growth or yield in the presence of an agricultural fieldapplication rate of a biocide.

26. The formulation of embodiment 25, wherein the biocontrol agentcomprises the strain deposited as NRRL No. B-50897 and the biocide isglyphosate.

27. The formulation of embodiment 25, wherein the biocontrol agentcomprises the strain AIP050999 deposited as NRRL No. B-50999 and thebiocide is glufosinate.

28. A method for improving the ability of a biocontrol agent to competein a field setting said method comprising modifying said biologicalagent such that said modified biocontrol agent is able to grow in thepresence of a herbicide.

29. A method for promoting plant growth, said method comprising applyinga composition comprising a modified population of biocontrol agents tothe soil where said plant is growing.

30. The method of embodiment 29, wherein said biocontrol agents havebeen modified to be resistant to glyphosate or glufosinate.

31. A method for growing a plant comprising applying to a crop, a seedor an area of cultivation a combination of an effective amount of abiocide and an effective amount of a modified biocontrol agent wherein

-   -   (a) the effective amount of the biocide is such as to        selectively control an organism of interest while the crop is        not significantly damaged; and,    -   (b) the effective amount of the modified biocontrol agent is        sufficient to result in a statistically significant increase in        plant health, yield and/or growth when compared to the plant        health, yield and/or growth that occurs when the same        concentration of a non-modified biocontrol agent is applied in        combination with the effective amount of the biocide.

32. The method of embodiment 31, wherein the modified biocontrol agentand the biocide are applied simultaneously.

33. The method of embodiment 32, wherein the modified biocontrol agentand the biocide are applied sequentially.

34. The method of any one of embodiments 31-33, wherein said biocontrolagent comprises the strain deposited as NRRL No. B-50897.

35. The method of embodiment 34, where the biocide is glyphosate, andwherein the effective amount of glyphosate is such as to selectivelycontrol weeds while the crop is not significantly damaged.

36. The method of any one of embodiments 31-33, wherein said biocontrolagent comprises the strain AIP050999 deposited as NRRL No. B-50999 andthe biocide is glufosinate.

37. The method of embodiment 36, where the biocide is glufosinate, andwherein the effective amount of glufosinate is such as to selectivelycontrol weeds while the crop is not significantly damaged.

38. A cultured population of a biocontrol agent wherein said culturedpopulation is produced by growing a population of agents underherbicide, fungicide, pesticide or a crop protection chemical pressureto select a purified culture of biocontrol agents that are resistant tosaid herbicide, fungicide, pesticide or other crop protection chemical.

39. The cultured population of the biocontrol agent of embodiment 38,wherein said biocontrol agent is present in an effective amountsufficient to improve plant health, growth or yield in the presence ofan agricultural field application rate of a biocide.

40. An isolated biologically pure culture of a biocontrol agent whereinsaid biocontrol agent is resistant to a biocide selected from aherbicide, a fungicide, a pesticide or a crop protection chemicalwherein said culture is produced by growing in the presence of saidbiocide.

41. The isolated biologically pure culture of the biocontrol agent ofembodiment 40, wherein said biocontrol agent is present in an effectiveamount sufficient to improve plant health, growth or yield in thepresence of an agricultural field application rate of a biocide.

42. The method of embodiment 38, wherein said composition comprises asuitable carrier.

43. A bacterial culture grown from the strain deposited as NRRL No.B-50897, wherein said bacterial culture has antifungal activity and isable to grow in the presence of glyphosate.

44. The bacterial culture of claim 43, wherein the strain deposited asNRRL No. B-50897 is present in an effective amount sufficient to improveplant health, growth or yield in the presence of an agricultural fieldapplication rate of glyphosate.

45. A bacterial culture grown from the strain AIP050999 deposited asNRRL No. B-50999, wherein said bacterial culture has antifungal activityand is able to grow in the presence of glufosinate.

46. The bacterial culture of claim 45, wherein the strain AIP050999deposited as NRRL No. B-50999 is present in an effective amountsufficient to improve plant health, growth or yield in the presence ofan agricultural field application rate of glufosinate.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 provides a growth curve of the various strains in the presence ofglyphosate.

DETAILED DESCRIPTION

Compositions and methods for improving biological control agents areprovided. A biological agent or biocontrol agent for purposes of thepresent invention is used to describe a microorganism that is used tocontrol disease-causing plant pathogens and plant pests. The biologicalcontrol agents of the invention have been modified such that they areable to grow in the presence of at least one biocide. A biocide is achemical substance which can exert a controlling effect on an organismby chemical or biological means. Biocides include pesticides, such asfungicides; herbicides; insecticides, other crop protection chemicals,and the like. Compositions of the invention include one or more isolatedbiocontrol agents that has been selected for resistance to biocides suchas a herbicide, fungicide, pesticide, or other crop protection chemical;a recombinant biocontrol agent that has been transformed to contain aherbicide, fungicide, pesticide, or other crop protection chemicalresistant gene; a modified population of biocontrol agents wherein thepopulation is resistant to at least one herbicide, fungicide, pesticide,or other crop protection chemical; and compositions comprising thesemodified populations of biocontrol agents. The modified population maycomprise microorganisms that have been selected for herbicide,fungicide, pesticide, or other crop protection chemical resistance orhave been transformed with a gene that confers resistance or toleranceto such herbicide, fungicide, pesticide, or other crop protectionchemical. Thus, the invention comprises substantially pure cultures, orbiologically pure cultures, of such modified biocontrol agents ormodified biological agents. A “biologically pure bacterial culture”refers to a culture of bacteria containing no other bacterial species inquantities to be detected by normal bacteriological techniques. Statedanother way, it is a culture wherein virtually all of the bacterialcells present are of the selected strain. A modified biocontrol agentincludes biocontrol agents that have acquired a trait due to selectionpressure and recombinant biocontrol agents that have been transformedwith a gene that confers resistance or tolerance to at least oneherbicide, fungicide, pesticide, or other crop protection chemical.

The invention further encompasses a particular modified biologicalcontrol agent. Such agent includes AIP1620. AIP1620 is a Pseudomonasstrain that has been selected for glyphosate tolerance. Additionalagents include AIP050999. AIP050999 is a Pseudomonas strain that hasbeen selected for glufosinate tolerance.

AIP1620 was deposited with the Patent Depository of the National Centerfor Agricultural Utilization Research Agricultural Research Service,U.S. Department of Agriculture, 1815 North University Street, Peoria,Ill. 61604 U.S.A. on Jan. 31, 2014 and assigned NRRL No. B-50897.AIP050999 was deposited with the Patent Depository of the NationalCenter for Agricultural Utilization Research Agricultural ResearchService, U.S. Department of Agriculture, 1815 North University Street,Peoria, Ill. 61604 U.S.A. on Jan. 23, 2015 and assigned NRRL No.B-50999. Each of these deposits will be maintained under the terms ofthe Budapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. This deposit wasmade merely as a convenience for those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. § 112.

By “herbicide, fungicide, pesticide, or other crop protection chemicaltolerance or herbicide, fungicide, pesticide, or other crop protectionchemical resistance” is intended the ability of an organism (i.e, theplant, the biocontrol agent, the biocontrol bacterial agent, etc.) tosurvive and reproduce following exposure to a dose of the herbicide,fungicide, pesticide, or other crop protection chemical that is normallylethal to the wild type organism.

Biological agents or biocontrol agents of the invention includemicroorganisms and fungi that control disease-causing plant pathogensand promote plant health, growth, and yield. Any of these biological orbiocontrol agents can be modified by selection or transformation andproduce a modified biological or biocontrol agent or recombinantbiological or biocontrol agent. Thus, the invention encompasses anisolated modified biocontrol agent. The modified biocontrol agents canbe grown to produce a population of biocontrol agents. By “modifiedpopulation of biological or biocontrol agents” is intended a populationof agents that substantially comprises a culture of the selected agentor the recombinant agent having the trait of interest such as resistanceto a herbicide, fungicide, pesticide, or other crop protection chemical.By substantially comprises is intended that the population has beengrown and produced from the modified or the recombinant biocontrolagent. That is, the modified or recombinant biocontrol agents can begrown to produce a biologically pure culture. It is recognized that suchbiologically pure cultures can be used together to enhance plant health,growth, or yield.

Any biological or biocontrol agent can be used in the methods of theinvention. Particular microorganisms of interest include strains of thebacteria Pseudomonas, Bacillus, Agrobacterium, Lysobacter, Gliocladium,Pythium, Chromobacterium, Penicillium, Pantoea, Lactobacillus,Paenibacillus, Burkholderia, Streptomyces, Variovorax, Pasteuria,Xanthomonas, etc. Fungi of interest include Aureobasidium, Ampelomyces,Beauveria, Metarhizium, Metschnikowia, Myrothecium, Lecanicillium,Chaetomium, Cordyceps, Coniothyrium, Dactylella, Gliocladium,Aspergillis, Paecilomyces, Trichoderma, Pisolithus, Glomus, etc. See,for example, U.S. Pat. Nos. 5,348,742; 5,496,547; 5,756,087; 5,955,348;6,060,051; 6,635,425; and U.S. Patent Publication 20130142759; all ofwhich are herein incorporated by reference. Many biocontrol agents areon the market and any of them can be modified according to the presentinvention. Such agents include: Agrobacterium radiobacter K84;Trichoderma atroviride; Bacillus subtilis GB03; Bacillus firmus I-1582;Trichoderma asperellum (ICC 012); T. gamsii (ICC 080); Bacillus pumilusstrain QST 2808; Bacillus subtilis strain QST 713; B. subtilis strainMBI 600; Paecilomyces furnosoroseus; Gliocladium catenulatum;Trichoderma harzianum rifai strain KRL-AG2; Trichoderma harzianum T-22;Trichoderma harzianum T-22; Trichoderma virens strain G-41; Trichodermaharzianum T-22; Bacillus subtilis QST 713; Bacillus amyloliquefaciensstrain D747; Trichoderma (Gliocladium) virens GL-21; Paecilomyceslilacinus; Paecilomyces fumosoroseus; Ampelomyces quisqualis; B.subtilis DSM 17231; B. licheniformis DSM 17236; Pythium oligandrum DV74; Bacillus subtilis GB03; Trichoderma asperellum; T. gamsii;Pseudomonas syringae ESC-10; Metschnikowia fructicola; Trichodermaharzianum T-22; Pseudomonas chlororaphis MA 342; B. amyloliquifaciens;Chrombacterium subtsugae strain PRAA4-1; B. subtilis amyloliquefaciensFZB24; Penicillium bilaii; Paecilomyces fumosoroseus FE 9901;Streptomyces lydicus WYEC 108; P. syringae A506; Coniothyrium minitans;Paecilomyces lilacinus strain 251; Streptomyces lydicus WYEC-108;Bacillus amyloliquifaciens; Trichoderma virens; Trichoderma viride;Ampelomyces quisqualis; Chaetomium globosum; Pseudomonas fluorescens;Bacillus subtilis; Bacillus pumulis; Myrothecium verrucaria AARC-0255;Streptomyces actinobacterium strain K61; Gliocladium catenulatum J1446;Aureobasidium pullulans strain DSM 14940; and A. pullulans strain DSM14941. Additional biological disease control products can be found onthe world wide web at:nevegetable.org/table-22-biological-disease-control-products.

Disease causing pathogens include fungi, bacteria, viruses andnematodes. Biocontrol agents of the invention are those that target anyof the plant pathogens. Target pathogens include but are not limited toAlternaria, Botrytis, Fusarium, Erwinia, Pseudomonas, Xanthomonas,Cercospora, Colletotrichum, Cladosporium, -Erisyphae spp., Microsphaerasyringae, Peronospora spp., Plasmopara spp., Phytophthora, Pythium,Rhizoctonia, Diplocarpon, Venturia, Mycosphaerella, Phomopsis, Taphrina,Elsinoe, Sclerotinia, Verticillum, Gnomonia, Fusicladium, Nectria,Phyllosticta, Diplocarpon, Albugo, Guignardia, Botrytis, Exobasidium,Entomosporium, Exobasidium, Pestalotia, Phoma, Cristulariella,Phakopsora, Thelaviopsis, Puccinia, Peronospora, Bremia, Pantoea,Clavibacter.

Herbicide, fungicide, pesticide, or other crop protection chemicalresistance is the ability of an organism to survive and reproducefollowing exposure to a dose of the herbicide, fungicide, pesticide, orother crop protection chemical that would normally be lethal to the wildtype organism or would substantially reduce growth of the wild typeorganism. Resistance may be induced or identified due to selection or itmay be induced through genetic engineering. To identify and produce amodified population of biocontrol agents through selection, thebiocontrol agents are grown in the presence of the herbicide, fungicide,pesticide, or other crop protection chemical as the selection pressure.Susceptible agents are killed while resistant agents survive toreproduce without competition. As the biocontrol agents are grown in thepresence of the herbicide, fungicide, pesticide, or other cropprotection chemical, resistant biocontrol agents successfully reproduceand become dominant in the population, becoming a modified population ofbiocontrol agents. Methods for selecting resistant strains are known andinclude U.S. Pat. Nos. 4,306,027 and 4,094,097, herein incorporated byreference. Therefore, the invention includes a biologically pure cultureof a resistant biocontrol strain. The resistant strains of the inventionhave the same identification characteristics as the original sensitivestrain except they are significantly more tolerant to the particularherbicide, fungicide, pesticide, or other crop protection chemical.Thus, their identification is readily possible by comparison withcharacteristics of the known sensitive strain.

Herbicides include glyphosate, ACCase inhibitors (Arloxyphenoxypropionate (FOPS)); ALS inhibitors (Sulfonylurea (SU)), Imidazonlinone(IMI), Pyrimidines (PM)); microtubule protein inhibitor (Dinitroaniline(DNA)); synthetic auxins (Phenoxy (P)), Benzoic Acid (BA), Carboxylicacid (CA)); Photosystem II inhibitor (Triazine (TZ)), Triazinone (TN),Nitriles (NT), Benzothiadiazinones (BZ), Ureas (US)); EPSP Synthaseinhibitor (glycines (GC)); Glutamine Synthesis inhibitor (PhosphinicAcid (PA)); DOXP synthase inhibitor (Isoxazolidinone (IA)); HPPDinhibitor (Pyrazole (PA)), Triketone (TE)); PPO inhibitors(Diphenylether (DE), N-phenylphthalimide (NP) (Ary triazinone (AT));VLFA inhibitors (chloroacetamide (CA)), Oxyacetamide (OA)); PhotosystemI inhibitor (Bipyridyliums (BP)); and the like.

Pesticides include imidacloprid clothianidin, arylpyrazole compounds(WO2007103076); organophosphates, phenyl pyrazole, pyrethoidscaramoyloximes, pyrazoles, amidines, halogenated hydrocarbons,carbamates and derivatives thereof, terbufos, chloropyrifos, fipronil,chlorethoxyfos, telfuthrin, carbofuran, imidacloprid, tebupirimfos (U.S.Pat. No. 5,849,320).

Fungicides include aliphatic nitrogen fungicides (butylamine, cymoxanil,dodicin, dodine, guazatine, iminoctadine); amide fungicides(benzovindiflupyr, carpropamid, chloraniformethan, cyflufenamid,diclocymet, diclocymet, dimoxystrobin, fenaminstrobin, fenoxanil,flumetover, furametpyr, isofetamid, isopyrazam, mandestrobin,mandipropamid, metominostrobin, orysastrobin, penthiopyrad, prochloraz,quinazamid, silthiofam, triforine); acylamino acid fungicides(benalaxyl, benalaxyl-M, furalaxyl, metalaxyl, metalaxyl-M, pefurazoate,valifenalate); anilide fungicides (benalaxyl, benalaxyl-M, bixafen,boscalid, carboxin, fenhexamid, fluxapyroxad, isotianil, metalaxyl,metalaxyl-M, metsulfovax, ofurace, oxadixyl, oxycarboxin, penflufen,pyracarbolid, sedaxane, thifluzamide, tiadinil, vanguard); benzanilidefungicides (benodanil, flutolanil, mebenil, mepronil, salicylanilide,tecloftalam); furanilide fungicides (fenfuram, furalaxyl, furcarbanil,methfuroxam); sulfonanilide fungicides (flusulfamide); benzamidefungicides (benzohydroxamic acid, fluopicolide, fluopyram, tioxymid,trichlamide, zarilamid, zoxamide); furamide fungicides (cyclafuramid,furmecyclox); phenylsulfamide fungicides (dichlofluanid, tolylfluanid);sulfonamide fungicides (amisulbrom, cyazofamid); valinamide fungicides(benthiavalicarb, iprovalicarb); antibiotic fungicides (aureofungin,blasticidin-S, cycloheximide, griseofulvin, kasugamycin, moroxydine,natamycin, polyoxins, polyoxorim, streptomycin, validamycin);strobilurin fungicides (fluoxastrobin, mandestrobin); methoxyacrylatestrobilurin fungicides (azoxystrobin, bifujunzhi, coumoxystrobin,enoxastrobin, flufenoxystrobin, jiaxiangjunzhi, picoxystrobin,pyraoxystrobin); methoxycarbanilate strobilurin fungicides(pyraclostrobin, pyrametostrobin, triclopyricarb); methoxyiminoacetamidestrobilurin fungicides (dimoxystrobin, fenaminstrobin, metominostrobin,orysastrobin); methoxyiminoacetate strobilurin fungicides(kresoxim-methyl, trifloxystrobin); aromatic fungicides (biphenyl,chlorodinitronaphthalenes, chloroneb, chlorothalonil, cresol, dicloran,fenjuntong, hexachlorobenzene, pentachlorophenol, quintozene, sodiumpentachlorophenoxide, tecnazene, trichlorotrinitrobenzenes); arsenicalfungicides (asomate, urbacide); aryl phenyl ketone fungicides(metrafenone, pyriofenone); benzimidazole fungicides (albendazole,benomyl, carbendazim, chlorfenazole, cypendazole, debacarb,fuberidazole, mecarbinzid, rabenzazole, thiabendazole); benzimidazoleprecursor fungicides (furophanate, thiophanate, thiophanate-methyl);benzothiazole fungicides (bentaluron, benthiavalicarb, benthiazole,chlobenthiazone, probenazole); botanical fungicides (allicin, berberine,carvacrol, carvone, osthol, sanguinarine, santonin); bridged diphenylfungicides (bithionol, dichlorophen, diphenylamine, hexachlorophene,parinol); carbamate fungicides (benthiavalicarb, furophanate, iodocarb,iprovalicarb, picarbutrazox, propamocarb, pyribencarb, thiophanate,thiophanate-methyl, tolprocarb); benzimidazolylcarbamate fungicides(albendazole, benomyl, carbendazim, cypendazole, debacarb, mecarbinzid);carbanilate fungicides (diethofencarb, pyraclostrobin, pyrametostrobin,triclopyricarb); conazole fungicides, conazole fungicides (imidazoles)(climbazole, clotrimazole, imazalil, oxpoconazole, prochloraz,triflumizole); conazole fungicides (triazoles) (azaconazole,bromuconazole, cyproconazole, diclobutrazol, difenoconazole,diniconazole, diniconazole-M, epoxiconazole, etaconazole, fenbuconazole,fluquinconazole, flusilazole, flutriafol, furconazole, furconazole-cis,hexaconazole, imibenconazole, ipconazole, metconazole, myclobutanil,penconazole, propiconazole, prothioconazole, quinconazole, simeconazole,tebuconazole, tetraconazole, triadimefon, triadimenol, triticonazole,uniconazole, uniconazole-P); copper fungicides (acypetacs-copper,Bordeaux mixture, Burgundy mixture, Cheshunt mixture, copper acetate,copper carbonate, basic, copper hydroxide, copper naphthenate, copperoleate, copper oxychloride, copper silicate, copper sulfate, coppersulfate, basic, copper zinc chromate, cufraneb, cuprobam, cuprous oxide,mancopper, oxine-copper, saisentong, thiodiazole-copper); cyanoacrylatefungicides (benzamacril, phenamacril); dicarboximide fungicides(famoxadone, fluoroimide); dichlorophenyl dicarboximide fungicides(chlozolinate, dichlozoline, iprodione, isovaledione, myclozolin,procymidone, vinclozolin); phthalimide fungicides (captafol, captan,ditalimfos, folpet, thiochlorfenphim); dinitrophenol fungicides(binapacryl, dinobuton, dinocap, dinocap-4, dinocap-6, meptyldinocap,dinocton, dinopenton, dinosulfon, dinoterbon, DNOC); dithiocarbamatefungicides (amobam, asomate, azithiram, carbamorph, cufraneb, cuprobam,disulfiram, ferbam, metam, nabam, tecoram, thiram, urbacide, ziram);cyclic dithiocarbamate fungicides (dazomet, etem, milneb); polymericdithiocarbamate fungicides (mancopper, mancozeb, maneb, metiram,polycarbamate, propineb, zineb); dithiolane fungicides (isoprothiolane,saijunmao); fumigant fungicides (carbon disulfide, cyanogen,dithioether, methyl bromide, methyl iodide, sodium tetrathiocarbonate);hydrazide fungicides (benquinox, saijunmao); imidazole fungicides(cyazofamid, fenamidone, fenapanil, glyodin, iprodione, isovaledione,pefurazoate, triazoxide); conazole fungicides (imidazoles) (climbazole,clotrimazole, imazalil, oxpoconazole, prochloraz, triflumizole);inorganic fungicides (potassium azide, potassium thiocyanate, sodiumazide, sulfur, see also copper fungicides, see also inorganic mercuryfungicides); mercury fungicides; inorganic mercury fungicides (mercuricchloride, mercuric oxide, mercurous chloride); organomercury fungicides((3-ethoxypropyl)mercury bromide, ethylmercury acetate, ethylmercurybromide, ethylmercury chloride, ethylmercury 2,3-dihydroxypropylmercaptide, ethylmercury phosphate,N-(ethylmercury)-p-toluenesulphonanilide, hydrargaphen,2-methoxyethylmercury chloride, methylmercury benzoate, methylmercurydicyandiamide, methylmercury pentachlorophenoxide,8-phenylmercurioxyquinoline, phenylmercuriurea, phenylmercury acetate,phenylmercury chloride, phenylmercury derivative of pyrocatechol,phenylmercury nitrate, phenylmercury salicylate, thiomersal,tolylmercury acetate); morpholine fungicides (aldimorph, benzamorf,carbamorph, dimethomorph, dodemorph, fenpropimorph, flumorph,tridemorph); organophosphorus fungicides (ampropylfos, ditalimfos, EBP,edifenphos, fosetyl, hexylthiofos, inezin, iprobenfos, izopamfos,kejunlin, phosdiphen, pyrazophos, tolclofos-methyl, triamiphos);organotin fungicides (decafentin, fentin, tributyltin oxide); oxathiinfungicides (carboxin, oxycarboxin); oxazole fungicides (chlozolinate,dichlozoline, drazoxolon, famoxadone, hymexazol, metazoxolon,myclozolin, oxadixyl, oxathiapiprolin, pyrisoxazole, vinclozolin);polysulfide fungicides (barium polysulfide, calcium polysulfide,potassium polysulfide, sodium polysulfide); pyrazole fungicides(benzovindiflupyr, bixafen, fenpyrazamine, fluxapyroxad, furametpyr,isopyrazam, oxathiapiprolin, penflufen, penthiopyrad, pyraclostrobin,pyrametostrobin, pyraoxystrobin, rabenzazole, sedaxane); pyridinefungicides (boscalid, buthiobate, dipyrithione, fluazinam, fluopicolide,fluopyram, parinol, picarbutrazox, pyribencarb, pyridinitril, pyrifenox,pyrisoxazole, pyroxychlor, pyroxyfur, triclopyricarb); pyrimidinefungicides (bupirimate, diflumetorim, dimethirimol, ethirimol,fenarimol, ferimzone, nuarimol, triarimol); anilinopyrimidine fungicides(cyprodinil, mepanipyrim, pyrimethanil); pyrrole fungicides(dimetachlone, fenpiclonil, fludioxonil, fluoroimide); quaternaryammonium fungicides (berberine, sanguinarine); quinoline fungicides(ethoxyquin, halacrinate, 8-hydroxyquinoline sulfate, quinacetol,quinoxyfen, tebufloquin); quinone fungicides (chloranil, dichlone,dithianon); quinoxaline fungicides (chinomethionat, chlorquinox,thioquinox); thiadiazole fungicides (etridiazole, saisentong,thiodiazole-copper, zinc thiazole); thiazole fungicides (ethaboxam,isotianil, metsulfovax, octhilinone, oxathiapiprolin, thiabendazole,thifluzamide); thiazolidine fungicides (flutianil, thiadifluor);thiocarbamate fungicides (methasulfocarb, prothiocarb); thiophenefungicides (ethaboxam, isofetamid, silthiofam); triazine fungicides(anilazine); triazole fungicides (amisulbrom, bitertanol, fluotrimazole,triazbutil); conazole fungicides (triazoles) (azaconazole,bromuconazole, cyproconazole, diclobutrazol, difenoconazole,diniconazole, diniconazole-M, epoxiconazole, etaconazole, fenbuconazole,fluquinconazole, flusilazole, flutriafol, furconazole, furconazole-cis,hexaconazole, huanjunzuo, imibenconazole, ipconazole, metconazole,myclobutanil, penconazole, propiconazole, prothioconazole, quinconazole,simeconazole, tebuconazole, tetraconazole, triadimefon, triadimenol,triticonazole, uniconazole, uniconazole-P); triazolopyrimidinefungicides (ametoctradin); urea fungicides (bentaluron, pencycuron,quinazamid); zinc fungicides (acypetacs-zinc, copper zinc chromate,cufraneb, mancozeb, metiram, polycarbamate, polyoxorim-zinc, propineb,zinc naphthenate, zinc thiazole, zinc trichlorophenoxide, zineb, ziram);unclassified fungicides (acibenzolar, acypetacs, allyl alcohol,benzalkonium chloride, bethoxazin, bromothalonil, chitosan,chloropicrin, DBCP, dehydroacetic acid, diclomezine, diethylpyrocarbonate, ethylicin, fenaminosulf, fenitropan, fenpropidin,formaldehyde, furfural, hexachiorobutadiene, methyl isothiocyanate,nitrostyrene, nitrothal-isopropyl, OCH, pentachlorophenyl laurate,2-phenylphenol, phthalide, piperalin, propamidine, proquinazid,pyroquilon, sodium orthophenylphenoxide, spiroxamine, sultropen,thicyofen, tricyclazole) or mefenoxam.

As indicated, recombinant biocontrol agents having resistance to aherbicide, fungicide, pesticide, or other crop protection chemical canbe made through genetic engineering techniques and such engineered orrecombinant biocontrol agents grown to produce a modified population ofbiocontrol agents. A recombinant biocontrol agent is produced byintroducing polynucleotides into the biocontrol host cell bytransformation. Methods for transforming microorganisms are known andavailable in the art. See, generally, Hanahan, D. (1983) Studies ontransformation of Escherichia coli with plasmids J. Mol. Biol. 166,557-77; Seidman, C. E. (1994) In: Current Protocols in MolecularBiology, Ausubel, F. M. et al. eds., John Wiley and Sons, NY; Choi etal. (2006) J. Microbiol. Methods 64:391-397; Wang et al. 2010. J. Chem.Technol. Biotechnol. 85:775-778. Transformation may occur by naturaluptake of naked DNA by competent cells from their environment in thelaboratory. Alternatively, cells can be made competent by exposure todivalent cations under cold conditions, by electroporation, by exposureto polyethylene glycol, by treatment with fibrous nanoparticles, orother methods well known in the art.

Herbicide resistance genes for use in transforming a recombinantbiocontrol agent include, but are not limited to, fumonisindetoxification genes (U.S. Pat. No. 5,792,931); acetolactate synthase(ALS) mutants that lead to herbicide resistance, in particular thesulfonylurea-type herbicides, such as the S4 and/or Hra mutations;inhibitors of glutamine synthase such as phosphinothricin or basta(e.g., bar gene); and glyphosate resistance (EPSPS gene)); and HPPDresistance (WO 96/38576, U.S. Pat. Nos. 6,758,044; 7,250,561; 7,935,869;and 8,124,846), or other such genes known in the art. The disclosures ofwhich are herein incorporated by reference. The bar gene encodesresistance to the herbicide basta, the nptll gene encodes resistance tothe antibiotics kanamycin and geneticin, and the ALS-gene mutants encoderesistance to the sulfonylurea herbicides including chlorsulfuron,metsulfuron, sulfometuron, nicosulfuron, rimsulfuron, flazasulfuron,sulfosulfuron, and triasulfuron, and the imadizolinone herbicidesincluding imazethapyr, imazaquin, imazapyr, and imazamethabenz.

The modified populations of biological control agents of the inventioncan be formulated as wettable powders, dusts, granules, aqueous or oilbased liquid products, and the like. Such formulations will comprise themodified biological control agents in addition to carriers and otheragents. The formulations can be used as field inoculants for biocontrol,seed coatings, etc. That is, the modified biocontrol populations can beused in any manner known in the art, including coating seeds with aneffective amount of the modified agents, in furrow application of themodified biocontrol populations directly into the soil, in foliarapplication, mixing into a potting mixture, and in post-harvest diseasecontrol. Such methods are known in the art and are described, forexample, in U.S. Pat. No. 5,348,742 and in published EuropeanApplication EP0472494 A2, both of which are herein incorporated byreference. Biocontrol includes management of resident populations oforganisms and introductions of specific organisms to reduce disease.

The biocontrol agent provided herein can be mixed with a fungicide,insecticide, or herbicide to enhance its activity or the activity of thechemical to which it has been added. In some cases the combination ofthe biocontrol agent and chemical may show synergistic activity, wherethe mixture of the two exceeds that expected from their simple additiveeffect.

The modified biological control agents of the invention can be used tosignificantly reduce disease, to promote plant growth and yield, and toreduce reliance on traditional pesticides. The modified agents of theinvention can be used with other pesticides for an effective integratedpest management program. In one embodiment, the modified biocontrolpopulations can be mixed in formulation with known pesticides in amanner described in WO 94/10845, herein incorporated by reference.

The modified biocontrol populations are applied in an effective amount.An effective amount of a modified biocontrol population is an amountsufficient to control or inhibit the pathogen. In other embodiments, theeffective amount of the modified biocontrol agent is an amountsufficient to promote or increase plant health, growth or yield in thepresence of an agricultural field application rate of a biocide. Therate of application of the modified biocontrol agent and/or the biocidemay vary according to the pathogen being targeted, the crop to beprotected, the efficacy of the modified biocontrol populations, theseverity of the disease, the climate conditions, and the like. Generallyfor a field inoculation, the rate of modified biocontrol agentapplication is 10¹² to 10¹⁶ colony forming units (CFU) per hectare.(This corresponds to about 10 g to 10 kg of active ingredient perhectare if the a.i. is 100 billion CFU per g.). In other embodiments,for a field inoculation, the rate of modified biocontrol agentapplication is 3×10¹⁵ to 1×10¹⁷ colony forming units (CFU) per hectare.(This corresponds to about 30 kg to 1000 kg of active ingredient perhectare if the a.i. is 100 billion CFU per g.). In other embodiments,for a field inoculation, the rate of modified biocontrol agentapplication is 3×10¹⁵ to 1×10¹⁷ colony forming units (CFU) per hectare;about 1×10¹² to about 1×10¹³ colony forming units (CFU) per hectare,about 1×10¹³ to about 1×10¹⁴ colony forming units (CFU) per hectare,about 1×10¹⁴ to about 1×10¹⁵ colony forming units (CFU) per hectare,about 1×10¹⁵ to about 1×10¹⁶ colony forming units (CFU) per hectare orabout 1×10¹⁶ to about 1×10¹⁷ colony forming units (CFU) per hectare. Inother embodiments, for a field inoculation, the rate of modifiedbiocontrol agent application is at least about 1×10¹³, about 1×10¹⁴,1×10¹⁵, about 1×10¹⁶ or about 1×10¹⁷ colony forming units (CFU) perhectare. In still other embodiments, the rate of modified biocontrolagent application is 10 g to 50 kg, 50 kg to 100 kg, 100 kg to 200 kg,200 kg to 300 kg, 300 kg to 400 kg, 400 kg, to 500 kg, 500 kg to 600 kg,600 kg to 700 kg, 700 kg to 800 kg, 800 kg to 900 kg, 900 kg to 1000 kgof active ingredient per hectare if the a.i. is 100 billion CFU per g.In still other embodiments, the rate of modified biocontrol agentapplication is at least 10 g, 50 kg, 100 kg, 200 kg, 300 kg, 400 kg, 500kg, 600 kg, 700 kg, 800 kg, 900 kg, 1000 kg of active ingredient perhectare if the a.i. is 100 billion CFU per g. In specific embodiments,the modified biocontrol agent applied comprises the strain deposited asNRRL No. B-50897 and/or the strain AIP050999 deposited as NRRL No.B-50999.

Any appropriate agricultural application rate for a biocide can beapplied to the crop, for example, an effective amount of the biocidethat controls a given organism (i.e., a pest of interest, such asfungus, insects, weeds, disease, etc) may be applied. Methods to assayfor the effective amount of the modified biocontrol agent include, forexample, any statistically significant increase in plant health, yieldand/or growth that occurs upon application of an effective amount of thebiocontrol agent and a field application rate of a biocide when comparedto the plant health, yield and/or growth that occurs when the sameconcentration of a non-modified biocontrol agent is applied incombination with the effective amount of the biocide.

Therefore, a further embodiment of the invention provides a method forcontrolling or inhibiting the growth of a plant pathogen by applying thepopulation of modified biological control agents of the invention to anenvironment in which the plant pathogen may grow. The application may beto the plant, to parts of the plant, to the seeds of the plants to beprotected, or to the soil in which the plant to be protected are growingor will grow. Application to the plant or plant parts may be before orafter harvest. Application to the seeds will be prior to planting of theseeds.

In other embodiments, a crop, area of cultivation, seed and/or weed canbe treated with a combination an effective amount of the modifiedcontrol agent and an effective amount of a biocide. By “treated with acombination of” or “applying a combination of” modified biocontrol agentand a biocide to a crop, area of cultivation or field it is intendedthat one or more of a particular field, plant crop, seed and/or weed istreated with one or more of the modified biocontrol agent and one ormore biocide so that a desired effect is achieved. Furthermore, theapplication of one or both of the modified biocontrol agent and thebiocide can occur prior to the planting of the crop (for example, to thesoil, the seed, or the plant). Moreover, the application of the modifiedbiocontrol agent and the biocide may be simultaneous or the applicationsmay be at different times (sequential), so long as the desired effect isachieved.

In one non-limiting embodiment, the modified biocontrol agent isresistant to glyphosate and further increases plant health, yield orgrowth when applied in an effective amount, and the biocide comprisesglyphosate or an active derivative thereof. In such methods, a seed,plant or area of cultivation is treated with a combination of aneffective amount of the modified biocontrol agent that is resistant toglyphosate and an effective amount of glyphosate, wherein the effectiveamount of glyphosate is such as to selectively control weeds while thecrop is not significantly damaged. In such embodiments, the effectiveamount of the modified biocontrol agent is sufficient to result in astatistically significant increase in plant health, yield and/or growthwhen compared to the plant health, yield and/or growth that occurs whenthe same concentration of a non-modified biocontrol agent is applied incombination with the effective amount of the glyphosate or activederivative thereof. In a further embodiment, the biocontrol agentcomprises an effective amount of AIP1620.

In another one non-limiting embodiment, the modified biocontrol agent isresistant to glufosinate and further increases plant health, yield orgrowth when applied in an effective amount, and the biocide comprisesglufosinate or an active derivative thereof. In such methods, a seed,plant or area of cultivation is treated with a combination of aneffective amount of the modified biocontrol agent that is resistant toglufosinate and an effective amount of glufosinate, wherein theeffective amount of glufosinate is such as to selectively control weedswhile the crop is not significantly damaged. In such embodiments, theeffective amount of the modified biocontrol agent is sufficient toresult in a statistically significant increase in plant health, yieldand/or growth when compared to the plant health, yield and/or growththat occurs when the same concentration of a non-modified biocontrolagent is applied in combination with the effective amount of theglufosinate or active derivative thereof. In a further embodiment, thebiocontrol agent comprises an effective amount of AIP050999.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which plants can be regenerated, plantcalli, plant clumps, and plant cells that are intact in plants or partsof plants such as embryos, pollen, ovules, seeds, leaves, flowers,branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips,anthers, and the like. Grain is intended to mean the mature seedproduced by commercial growers for purposes other than growing orreproducing the species. Progeny, variants, and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise the introduced polynucleotides.

The modified biocontrol agent can be employed with any plant species,including, but not limited to, monocots and dicots. Examples of plantspecies of interest include, but are not limited to, corn (Zea mays),Brassica sp. (e.g., B. napus, B. rapa, B. juncea), particularly thoseBrassica species useful as sources of seed oil, alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetumglaucum), proso millet (Panicum miliaceum), foxtail millet (Setariaitalica), finger millet (Eleusine coracana)), sunflower (Helianthusannuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum),soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense,Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihotesculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple(Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao),tea (Camellia sinensis), banana (Musa spp.), avocado (Persea americana),fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica),olive (Olea europaea), papaya (Carica papaya), cashew (Anacardiumoccidentale), macadamia (Macadamia integrifolia), almond (Prunusamygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.),oats, barley, vegetables, ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). In specific embodiments, plants of thepresent invention are crop plants (for example, corn, alfalfa,sunflower, Brassica, soybean, cotton, safflower, peanut, sorghum, wheat,millet, tobacco, etc.). In other embodiments, a corn or soybean plantsis employed.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL Example 1 Production of Glyphosate Resistant Mutants ofAIP0069

Introduction

Biological agents are now being used in agriculture to reduce risk andimprove yield. One important attribute of these biological agents isthat they must be compatible with chemicals that may also be applied incommercial farming practice. Glyphosate is a chemical herbicide thataccounts for about 25% of the global herbicide market and is applied ata rate of around 200 million pounds per year. This herbicide inhibitsthe enzyme, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EPSPS)which catalyzes one step in aromatic amino acid biosynthesis in plantsand many bacteria. Thus glyphosate decreases viability of organismsincluding any biocontrol agents that rely on EPSPS, and it has beenreported to alter the plant microbial community. This report describessuccessful introduction of glyphosate tolerance into a Pseudomonasfluorescens strain used in biological control of several importantfungal plant pathogens, including Pythium and Rhizoctonia, the causalagents in the agriculturally important “Damping Off” disease complex. Inaddition to improving chemical compatibility of this biocontrol agent,introduction of glyphosate resistance offers additional advantages incommercial production.

In addition to the example of glyphosate provided here, otheragricultural chemicals may inhibit the growth of desirable biologicalcontrol or plant growth promoting bacteria. Examples include theherbicides glufosinate (glutamine synthase inhibitor), sulfonylurea andimidazolinone herbicides (branched chain aminio acid synthesisinhibitors) and the antibiotics streptomycin, oxytetracycline andkasugamycin.

Materials and Methods and Results

The biological control strain Pseudomonas fluorescens AIP0069 wasstreaked onto agar plates containing 0 or 5 mM glyphosate. The basalmedium consisted of 11.3 g Na₂HPO₄.7H₂O, 3 g KH₂PO₄, 1 g NH₄Cl, 10 gmonosodium glutamate, 31 g molasses, 493 mg MgSO₄.7H₂O, 50 mgZnSO₄.7H₂O, 5 mg FeSO₄.7H₂O, and 0.3 g thiamine per liter of deionizedwater. In the absence of glyphosate numerous bacterial colonies werevisible after incubating overnight at 25 C. In the presence of 5 mMglyphosate no colonies were visible after a similar incubation, however,after extended incubation of several days a few colonies were seen.These colonies were isolated and grown in liquid medium at multipleglyphosate concentrations. One isolate, named GlyphR1, was ten-fold moreresistant to glyphosate than the parent AIP0069 strain (FIG. 1). Thisimproved strain is expected to be more competitive, and thus moreeffective as a biocontrol, than AIP0069 or similar glyphosate-sensitivestrains in agricultural systems where glyphosate is present in the soiland crops. Also, glyphosate can be used as a selective agent during theproduction, formulation and/or storage of this strain to preventcontamination by other bacteria.

Example 2 Biological Control Activity of Glyphosate Resistant Mutants

Bacteria were inoculated into 50 ml of broth medium consisting of 11.3 gNa₂HPO₄.7H₂O, 3 g KH₂PO₄, 1 g NH₄Cl, 10 g Monosodium glutamate, 30 gmolasses, 493 mg MgSO₄.7H₂O, 50 mg ZnSO₄.7H₂O, and 5 mg FeSO₄.7H₂O perliter of deionized water. Cultures were grown in 250 ml baffled flasksin a shaking incubator at 28 C, 250 rpm for 2 days. Cells were collectedby centrifugation at 3500×g for 10 minutes. The culture supernatantswere discarded and the cells were resuspended in sterile deionized waterto the volumes of the original cultures. AIP0323, a mutant of AIP0069which does not have antifungal activity, was included as a negativecontrol.

Rhizoctonia solani infested rice grain was produced as describedpreviously (K. A. Holmes and D. M. Benson, 1994. Evaluation ofPhytophthora parasitica var. nicotianae as a biocontrol for Phytophthoraparasitica on Catharanthus roseus. Plant Disease 78:193-199.) Infestedrice was pulverized in a blender and screened through a #10 sieve (2 mmopening). The pulverized grain was mixed with germination medium at therate of 2 g per liter.

Impatiens seeds were planted into the infested germination medium insize 402 plug trays, treated with 0.3 ml of resuspended bacteria perplug and grown under standard greenhouse production conditions. Therewere 2 replicates for each experimental treatment with 20 plugs perreplicate. The number of healthy seedlings was assessed after two weeks.The data below in Table 1 demonstrate that the glyphosate resistantvariants AIP0404 and AIP1620 retain full antifungal activity, comparedto the progenitor strain, AIP0069.

TABLE 1 % healthy seedlings Treatment Replicate 1 Replicate 2 AverageNoninoculated control 90 90 90 Inoculated control 15 20 17.5Inoculated + AIP0069 75 80 77.5 Inoculated + AIP0323 10 15 12.5Inoculated + AIP0404 75 80 77.5 Inoculated + AIP1620 80 75 77.5

The biocontrol strain can be used to control Fusarium head blight, AsianSoybean Rust, Rhizoctonia, Botrytis, Pythium, turf diseases, and thelike.

Example 3

Genes encoding glyphosate tolerant EPSPS enzymes may be obtained fromvarious bacteria (A. Schulz et al, 1985. Differential sensitivity ofbacterial 5-enolpyruvylshikimate-3-phosphate synthases to the herbicideglyphosate. FEMS Microbiology Letters, 28:297-301). In particular, theEPSPS genes from Agrobacterium tumefaciens CP4 (G. F. Barry et al, 1992.Inhibitors of amino acid biosynthesis: Strategies for impartingglyphosate tolerance to crop plants. p. 139-145. In B. K. Singh et al.(e.) Biosynthesis and molecular regulation of amino acids in plants. Am.Soc. Plant Physiologists, Rockville, Md.), and Arthrobacter globiformis(C. L. Peters et al, 2010, GRG23 and GRG51 genes conferring herbicideresistance. U.S. Pat. No. 7,674,958), are highly resistant.

A suitable gene is amplified by PCR or made synthetically usingtechniques well known in the art. The open reading frame is cloned intothe plasmid vector pKK223-3 (Pharmacia) between the tacI promoter andthe rrnB transcriptional terminator. The tad promoter provides strongconstitutive expression of genes in Pseudomonas. Genomic DNA sequencesfrom strain AIP0069 are incorporated on each side of thepromoter—gene—terminator cassette to direct homologous recombinationinto the AIP0069 chromosome.

The resulting plasmid is mobilized from E. coli to Pseudomonasfluorescens AIP0069 by conjugation, a technique well known in the art,and selection on defined medium containing 100 mM glyphosate. Theplasmid contains the narrow host range colE1 origin of replication andthus cannot replicate in Pseudomonas. Glyphosate resistant colonies willbe obtained when the promoter—gene—terminator cassette integrates intothe Pseudomonas chromosome by homologous recombination. Single crossoverevents (where the entire plasmid is integrated into the chromosome) aredistinguished from double crossover events (where only the desiredpromoter—gene—terminator cassette is integrated) by PCR, Southernblotting, or other techniques well known in the art. A double crossoverevent is selected for use.

Example 4

AIP1620 starter cultures were inoculated using colonies from Luria agarplates and grown in 0.1×NBY broth (0.8 g of Difco Nutrient Broth powderand 0.5 g of yeast extract powder per liter of deionized water) andgrown at 28 C, 250 rpm. Production cultures were grown in a brothcontaining, per liter of deionized water: 11.3 g Na₂HPO₄.7H₂O, 3.0 gKH₂PO₄, 1.0 g NH₄Cl, 10 g monosodium glutamate, 3.0 g molasses, 0.49 gMgSO₄.7H₂O, 50 mg ZnSO₄.7H₂O, 5 mg FeSO₄.7H₂O and sufficienthydrochloric acid to adjust the pH to approximately 6.2. Fifty ml ofproduction broth was placed in a 250 ml baffled culture flask,inoculated with 0.5 ml of starter culture and incubated at 28 C, 250rpm. The production cultures were inoculated at various times and thenharvested simultaneously to yield cultures with incubation times of 15,24, 33, and 43 hours. Forty ml of each culture was harvested bycentrifugation. The spent culture broth was discarded and the cells werere-suspended in autoclaved deionized water to 40 ml final volume.

Fungal inoculum was prepared using the rice grain method described byHolmes and Benson (K. A. Holmes and D. M. Benson, 1994. Evaluation ofPhytophthora parasitica var. nicotianae for biocontrol of Phytophthoraparasitica on Catharanthus roseus. Plant Disease, 78:193-199.). Infestedrice grains were pulverized in a blender and screened through a #10sieve. This inoculum was mixed into Fafard superfine germinating mix atthe rate of 1.0 g per liter.

The inoculated germination mix was placed in 392 greenhouse plug trays(Landmark Plastic Corporation, Akron, Ohio) and one impatiens seed wasplanted into each cell. AIP1620 cell suspensions were applied at therate of 0.3 ml per cell. The seeds were germinated under standardgreenhouse conditions. There were 3 replicates of each treatment with 20cells per replicate. After 10 to 14 days the assays were scored bycounting the number of healthy seedlings in each treatment. Results aresummarized in Table 2 below.

TABLE 2 Performance AIP1620 cells in greenhouse seed germination assayLive seedlings (out of 20) Culture Standard Treatment time Meandeviation Non-inoculated control n/a 15.0 0.0 Inoculated control n/a 2.71.2 AIP1620 15 hrs 6.3 1.2 AIP1620 24 hrs 13.3 4.6 AIP1620 33 hrs 15.00.0 AIP1620 44 hrs 14.7 2.5

Example 5

Multiple greenhouse trials of AIP1620 cells were performed over a 10month period. For each trial AIP1620 cultures were grown, harvested andre-suspended in autoclaved deionized water essentially as described inExample 4 using a culture time of approximately 24 hours. The greenhousegermination trials also were performed as described in Example 4, butthe R. solani inoculum rate varied from 0.25 to 1.0 g of pulverized ricegrain per liter of germination mix, depending on the trial. The resultscompiled from 17 trials are shown in Table 3 below and demonstrateconsistent performance of AIP1620 in controlling damping off disease.

TABLE 3 Performance AIP1620 cells in multiple greenhouse seedgermination assays Live seedlings (out of 20) Non- Inoculated inoculatedInoculated plus Control Control AIP1620 Trial Mean SD Mean SD Mean SD 117.0 1.0 1.3 0.6 11.3 2.5 2 17.3 2.5 9.0 1.0 17.3 2.1 3 14.0 0.0 2.0 1.016.7 2.3 4 12.3 1.5 1.7 1.5 13.3 3.1 5 18.4 1.1 3.8 1.8 16.8 1.3 6 16.41.7 2.6 2.1 13.6 2.5 7 16.6 1.1 2.8 1.3 15.8 1.3 8 19.7 0.6 3.0 1.0 17.30.6 9 19.0 1.0 3.7 1.5 15.7 1.2 10 16.3 0.6 1.7 2.1 15.7 0.6 11 18.5 0.72.0 0.0 14.5 0.7 12 18.0 1.0 3.0 3.0 16.3 0.6 13 18.8 1.9 3.8 2.1 17.00.8 14 17.0 0.0 3.5 0.7 15.5 0.7 15 18.6 1.1 4.8 1.3 14.8 2.8 16 19.50.6 3.5 2.5 18.5 1.5 17 18.0 0.0 1.0 1.7 15.3 0.6

Example 6

Fifty grams of AIP1620 cell paste was mixed with 50 g of Min-U-Gel 400or Min-U-Gel 200 attapulgite clay (Active Minerals International, LLC,Sparks, Md.) dried to a water activity of less than 0.3. One portion ofeach formulation was stored at 4° C. and another was stored at 22° C.The viability of these formulations was tested at various times bydilution plating and the results are shown in Table 4 below. After 21days in storage, the samples which had been stored at 4° C. were testedin a greenhouse seed germination assay and found to have retainedantifungal activity against Rhizoctonia solani.

TABLE 4 Survival of formulated AIP1620 cells during storage at 4° C. or22° C. Colony forming units per gram of AIP1620 after storage forFormulation 2 weeks 4 weeks 20 weeks Min-U-Gel 400 5.4 × 10⁷ 2.0 × 10⁶<2.0 × 10⁴  stored at 22° C. Min-U-Gel 400 1.3 × 10⁸ 3.5 × 10⁸ Nottested stored at 4° C. Min-U-Gel 200 8.2 × 10⁸ 3.7 × 10⁸ 7.2 × 10⁵stored at 22° C. Min-U-Gel 200  1.8 × 10¹⁰ 9.0 × 10⁹ Not tested storedat 4° C.

Example 7

One hundred grams of AIP1620 cell paste was mixed with 20 g of syntheticcalcium silicate (MicroCel E, Imerys Filtration Minerals, Lompoc,Calif.) using a food processor. The resulting material contained2.7×10¹⁰ colony forming units per gram (CFU/g) of AIP1620, as determinedby dilution plating. This material was dried at 40 C to a water activityof less than 0.30 at which time it contained 1.4×10⁹ CFU/g of AIP1620.The dried powder formulation was stored in vacuum sealed mylar pouchesat 22 C. After 85 days the powder contained 1.1×10⁶ CFU/g of AIP1620 andretained antifungal activity against Rhizoctonia solani as determined bya greenhouse seed germination assay.

Example 8

One hundred grams of AIP1620 cell paste was mixed with 5 g of glyceroland 20 g of synthetic calcium silicate using a food processor. Theresulting material contained 5.7×10¹¹ CFU/g of AIP1620, as determined bydilution plating. This material was dried at 40 C to a water activity ofless than 0.30 at which time it contained 3.1×10⁹ CFU/g of AIP1620. Thedried powder formulation was stored in vacuum sealed mylar pouches at 22C. After 61 days the powder contained 6.2×10⁸ CFU/g of AIP1620 andretained antifungal activity against Rhizoctonia solani as determined bya greenhouse seed germination assay.

Example 9

One hundred grams of AIP1620 cell paste was mixed with 5 g of trehaloseand 20 g of synthetic calcium silicate using a food processor. Theresulting material contained 5.7×10¹¹ CFU/g of AIP1620, as determined bydilution plating. This material was dried at 40 C to a water activity ofless than 0.30 at which time it contained 4.0×10⁸ CFU/g of AIP1620. Thedried powder formulation was stored in vacuum sealed mylar pouches at 22C. After 54 days the powder contained 2.7×10⁷ CFU/g of AIP1620.

Example 10

Four grams of xanthan gum and was dispersed into 4 g of soybean oil. Theresulting mixture was combined with 100 g of AIP1620 cell paste andallowed to thicken for about 5 minutes at room temperature. Thethickened mixture was blended into 20 g of synthetic calcium silicateusing a food processor. The resulting material contained 9.4×10¹¹ CFU/gof AIP1620 and was divided into two 50 g portions. One portion was driedat 40 C to a water activity of <0.30 at which time it contained 7.0×10⁸CFU/g of AIP1620.

The other portion was dried over silica gel at room temperature to awater activity of <0.10 at which time it contained 1.18×10¹⁰ CFU/g ofAIP1620.

Example 11

Five different formulations were prepared essentially as described inExample 4, above, using the excipients and proportions shown in Table 5,below. These formulations were dried at 40° C. to a water activity ofless than 0.30 and stored at 4° C.

Fungal inoculum was prepared using the rice grain method described byHolmes and Benson (K. A. Holmes and D. M. Benson, 1994. Evaluation ofPhytophthora parasitica var. nicotianae for biocontrol of Phytophthoraparasitica on Catharanthus roseus. Plant Disease, 78:193-199.). Infestedrice grains were pulverized in a blender and screened through a #10sieve. This inoculum was mixed into Fafard superfine germinating mix atthe rate of 0.25 g per liter.

The inoculated mix was divided and formulated AIP1620 was added at therate of 5 g per liter. Impatiens seed were planted into the inoculatedand treated mixes. The seeds were germinated under standard greenhouseconditions. After 10 days the assays were scored by counting the numberof healthy seedlings in each treatment. Results are summarized in Table6 below.

TABLE 5 Composition of formulations AIP1620 Final water Formulation cellpaste Excipient activity A 50 g 50 g Minugel 400 0.29 attapulgite clay B50 g 50 g Minugel 200 0.28 attapulgite clay C 50 g 50 g Agsorb 325 RVM0.24 attapulgite clay D 100 g 15 g Sipernat 22S 0.26 hydrophilic silicaE 100 g 15 g Aerosil 200F 0.29 silica

TABLE 6 Performance of formulated AIP1620 in greenhouse seed germinationassay Live seedlings (out of 20) Storage Standard Treatment time Meandeviation Non-inoculated control n/a 19.7 0.6 Inoculated control n/a 3.01.0 Formulation A 45 days 12.7 4.9 Formulation B 45 days 9.0 6.2Formulation C 45 days 17.3 1.2 Formulation D 45 days 17.3 0.6Formulation F 45 days 11.7 2.1

Example 12

Several formulations were prepared as described in Examples 4 through 8,above, at different times. The composition of the different formulationsis shown in Table 7, below. After drying to a water activity of 0.30 orlower, the formulated materials were vacuum sealed into mylar pouchesand stored at 22° C.

Fungal inoculum was prepared using the rice grain method described byHolmes and Benson (K. A. Holmes and D. M. Benson, 1994. Evaluation ofPhytophthora parasitica var. nicotianae for biocontrol of Phytophthoraparasitica on Catharanthus roseus. Plant Disease, 78:193-199.). Infestedrice grains were pulverized in a blender and screened through a #10sieve. This inoculum was mixed into Fafard superfine germinating mix atthe rate of 0.25 g per liter.

The inoculated mix was divided and formulated AIP1620 was added at therate of 5 g per liter. On the same day, a subsample of each formulationwas dilution plated to determine the CFU/g of AIP1620. Impatiens seedwere planted into the inoculated and treated mixes. The seeds weregerminated under standard greenhouse conditions. After 10 to 14 days theassays were scored by counting the number of healthy seedlings in eachtreatment. Results are summarized in Table 8 below.

TABLE 7 Composition of formulations Formula- AIP1620 Drying tion cellpaste Additions Excipient method F 100 g None 20 g calcium 40° C.silicate oven G 100 g 5 g glycerol 20 g calcium 40° C. silicate oven H100 g none 50 g MinuGel 40° C. 200 oven I 100 g 4 g xanthan gum, 20 gcalcium Room temp, 4 g soybean oil silicate silica gel J 100 g 4 gxanthan gum, 20 g calcium 40° C. 4 g soybean oil silicate oven K 100 g 4g xanthan gum, 20 g calcium 40° C. 4 g olive oil silicate oven

TABLE 8 Performance of formulated AIP1620 in greenhouse seed germinationassay CFU Live seedlings (out of 20) Storage AIP1620 Standard Treatmenttime per g Mean deviation Non-inoculated control n/a 18.3 0.6 Inoculatedcontrol n/a 2.3 3.2 Formulation F 12 days 5.2 × 10⁶ 5.7 2.5 FormulationG 12 days 8.3 × 10⁶ 10.3 3.2 Formulation F 18 days 8.0 × 10⁵ 16.7 1.5Formulation G 18 days 3.4 × 10⁹ 16.7 1.1 Formulation H 25 days 5.7 × 10⁹4.7 0.7 Formulation I 39 days 7.7 × 10⁹ 16.7 1.5 Formulation J 39 days1.3 × 10⁷ 8.0 1.7 Formulation K 39 days 3.6 × 10⁸ 5.3 1.5

These results demonstrate that formulated AIP1620 retains viability andactivity, that is, the ability to protect seedlings against damping offdisease.

Example 13

Fifty grams of AIP1620 cell paste was mixed with 50 g of Min-U-Gel 400or Min-U-Gel 200 attapulgite clay (Active Minerals International, LLC,Sparks, Md.) dried to a water activity of less than 0.2, and stored at22 C. The viability of these formulations was tested at various times bydilution plating and the results are shown in Table 9 below. After 21days both formulations were tested in a greenhouse seed germinationassay and found to have retained antifungal activity against Rhizoctoniasolani.

TABLE 9 Colony forming units per gram of AIP1620 after storage at 22° C.for Formulation 14 days 30 days 141 days A: Min-U-Gel 400 1.3 × 10⁶ 3.5× 10⁶ <2.0 × 10⁴  B: Min-U-Gel 200 1.8 × 10⁸ 9.0 × 10⁷ 7.2 × 10⁵

Example 14

Greenhouse experiments were performed to demonstrate the efficacy ofAIP1620 in controlling Botrytis cinerea.

AIP1620 starter cultures were inoculated using colonies from Luria agarplates and grown in 0.1×NBY broth (0.8 g of Difco Nutrient Broth powderand 0.5 g of yeast extract powder per liter of deionized water) andgrown at 28 C, 250 rpm. Production cultures were grown in a brothcontaining, per liter of deionized water: 11.3 g Na₂HPO₄.7H₂O, 3.0 gKH₂PO₄, 1.0 g NH₄Cl, 10 g monosodium glutamate, 3.0 g molasses, 0.49 gMgSO₄.7H₂O, 50 mg ZnSO₄.7H₂O, 5 mg FeSO₄.7H₂O and sufficienthydrochloric acid to adjust the pH to approximately 6.2. Fifty ml ofproduction broth was placed in a 250 ml baffled culture flask,inoculated with 0.5 ml of starter culture and incubated at 28 C, 250rpm. The production cultures were inoculated at various times and thenharvested simultaneously to yield cultures with incubation times of 15,24, 33, and 43 hours. Forty ml of each culture was harvested bycentrifugation. The spent culture broth was discarded and the cells werere-suspended in autoclaved deionized water to 40 ml final volume.

Botrytis cinerea was grown on potato dextrose broth for 1 to 2 weekswithout shaking. The resulting mycelial mat was removed from the brothand homogenized in autoclaved deionized water to produce liquidinoculum.

Organically grown strawberries were purchased at a local market. Theunblemished fruits were selected and dipped into the B. cinerea inoculumfor 2 to 3 seconds, then allowed to dry for 60 minutes before treatment.AIP1620 treatments were applied by dipping the inoculated fruits intothe cell suspension for 2-3 seconds. The fruit were then placed intosealed plastic containers with moist paper towels to maintain highhumidity and stored at room temperature for 72 to 84 hours. There were14 replicates (berries) in each treatment. Each berry was rated on avisual spoilage severity scale of 0=no damage, 1=25%, 2=50% damage,3=75% damage, and 4=100% (i.e., complete coverage of the berry by thefungus). Results are summarized in Table 10 below.

TABLE 10 Postharvest control of Grey Mold of strawberries by AIP1620cells Treatment Average spoilage severity rating Non-inoculated control3.1 Inoculated control 3.3 Inoculated plus AIP1620 0.7

Example 15

Greenhouse experiments were performed to demonstrate the efficacy ofAIP1620 in controlling damping off disease caused by the oomycete plantpathogen Pythium aphanadermatum.

AIP1620 starter cultures were inoculated using colonies from Luria agarplates and grown in 0.1×NBY broth (0.8 g of Difco Nutrient Broth powderand 0.5 g of yeast extract powder per liter of deionized water) andgrown at 28 C, 250 rpm. Production cultures were grown in a brothcontaining, per liter of deionized water: 11.3 g Na₂HPO₄.7H₂O, 3.0 gKH₂PO₄, 1.0 g NH₄Cl, 10 g monosodium glutamate, 3.0 g molasses, 0.49 gMgSO₄.7H₂O, 50 mg ZnSO₄.7H₂O, 5 mg FeSO₄.7H₂O and sufficienthydrochloric acid to adjust the pH to approximately 6.2. Fifty ml ofproduction broth was placed in a 250 ml baffled culture flask,inoculated with 0.5 ml of starter culture and incubated at 28 C, 250rpm. The production cultures were inoculated at various times and thenharvested simultaneously to yield cultures with incubation times of 15,24, 33, and 43 hours. Forty ml of each culture was harvested bycentrifugation. The spent culture broth was discarded and the cells werere-suspended in autoclaved deionized water to 40 ml final volume.

Inoculum of P. aphanadermatum was prepared using the rice grain methoddescribed by Holmes and Benson (K. A. Holmes and D. M. Benson, 1994.Evaluation of Phytophthora parasitica var. nicotianae for biocontrol ofPhytophthora parasitica on Catharanthus roseus. Plant Disease,78:193-199.). Infested rice grains were pulverized in a blender andscreened through a #10 sieve. This inoculum was mixed into Fafardsuperfine germinating mix at the rate of 6.0 g per liter (trials 1-4) or7.0 g per liter (trial 5).

The inoculated germination mix was placed in 392 greenhouse plug trays(Landmark Plastic Corporation, Akron, Ohio) and one impatiens seed wasplanted into each cell. AIP1620 cell suspensions were applied at therate of 0.3 ml per cell. The seeds were germinated under standardgreenhouse conditions. There were 2 or 3 replicates of each treatmentwith 20 cells per replicate. After 7 to 17 days the assays were scoredby counting the number of healthy seedlings in each treatment. Resultsare summarized in Table 11 below.

TABLE 11 Control of Pythium damping off disease by AIP1620 cells in agreenhouse seed germination assay. Values are mean +/− standarddeviation. Live seedlings (out of 20) Treatment Trial 1 Trial 2 Trial 3Trial 4 Trial 5 Non- 18.0 ± 1.4 18.5 ± 0.7 17.5 ± 0.7 16.5 ± 0.7 17.3 ±0.0 inoculated control Inoculated 12.0 ± 0.0 10.5 ± 0.7  9.0 ± 1.4  8.5± 0.7 10.7 ± 0.7 control Inoculated 17.0 ± 1.4 15.5 ± 2.1 13.5 ± 2.112.5 ± 0.7 16.3 ± 0.7 plus AIP1620

Example 16 Control of Asian Soybean Rust with AIP1620

AIP1620 cells were produced as described in the previous examples.Phakopsora pachyrhizi was grown on susceptible soybean plants andureidinospores were harvested by vacuum suction from infected leaveswhich manifested erupted pustules (Twizeyimana, M., and Hartman, G. L.2010. Culturing Phakopsora pachyrhizi on detached leaves andurediniospore survival at different temperatures and relativehumidities. Plant Disease 94:1453-1460).

Williams 82 soybean plants were grown in plant growth chambers usingtechniques well known in the art. When plants were at V3-stage, thefirst fully expanded trifoliate leaf was sprayed with re-suspendedAIP1620 cells, a chemical fungicide standard, or deionized water(inoculated control). One day later the leaves were inoculated with asuspension of P. pachyrhizi ureidinospores (1×10⁵/ml). Both inoculationand strain/fungicide were applied using an atomizer attached to an aircompressor. The plants were maintained in a growth chamber at 95% RHwith a daily cycle of 12 h of light and 12 h of darkness at 21 and 23°C., respectively. After two weeks disease severity was scored bycounting the number of sporulating uredinia in randomly selected 1 cmdiameter circles on the inoculated leaves. There were 3 replications(plants) for each treatment and 3 uredinia counts for each replication.The results are shown in table 12, below and demonstrate thatapplications of AIP1620 effectively control Asian Soybean Rust diseasecaused by Phakopsora pachyrhizi.

TABLE 12 Sporulating uredinia per 1-cm Treatment diameter circleInoculated control 22.1 Chemical standard 0.0 AIP1620 1.8

Example 17 AIP1620 Compatibility with Commercial Fungicides

The viability of AIP1620 was measured after mixing the strain 3commercial fungicides, each containing a different active ingredient(Table 13). Fungicide concentrations were selected to simulate those ina typical tank mix for field application.

AIP1620 was grown in 3 mL of LB medium in a 10 mL tube for 24 hours at28° C., 250 rpm. Cell pellets were harvested by centrifugation andsuspended in 3 mL of dH₂0. Nine hundred microliters of cell suspensionwas mixed with 100 microliters of 10× fungicide stock and incubated at28° C. for 5 minutes or 120 minutes.

TABLE 13 Formulated chemical fungicides Brand Name Active ingredientVolume of product per 100 mL) Quadris Azoxystrobin 936 μL SpectatorPropiconazole 1.87 mL Ultra 1.3 Subdue Maxx Mefenoxam 78.1 μL

After incubation with the fungicides the cells were harvested bycentrifugation as described above, re-suspended in deionized water.Aliquots were serially diluted in deionized water, plated on LB agar andincubated at 28 C for 2 days using techniques well known in the art.

Bacterial colonies were counted and the number of colony forming unitsper ml (CFU/ml) in the original solutions were calculated. The data areshown in Table 14 below and demonstrate that the viability of AIP1620 isnot adversely affected by mixing with these formulated fungicides.

TABLE 14 Viability of AIP1620 cells after incubation with fungicideformulations in a simulated tank mix. CFU/mL after incubation timeFungicide 5 Minutes 120 Minutes Water (control) 3.70 × 10⁹ 8.67 × 10⁸Quadris 4.50 × 10⁹ 4.87 × 10⁹ Spectator 2.33 × 10⁹ 2.17 × 10⁹ Subdue4.67 × 10⁹ 3.63 × 10⁹

Example 18 Evaluation of the Protectant Activity of Mixtures of AIP1620and Fungicides Against Asian Soybean Rust Caused by Phakopsorapachyrhizi

Bacteria are inoculated into 50 ml of broth medium consisting of 11.3 gNa₂HPO₄.7H₂O, 3 g KH₂PO₄, 1 g NH₄Cl, 10 g Monosodium glutamate, 30 gmolasses, 493 mg MgSO₄.7H₂O, 50 mg ZnSO₄.7H₂O, and 5 mg FeSO₄.7H₂O perliter of deionized water. Cultures are grown in 250 ml baffled flasks ina shaking incubator at 28 C, 250 rpm for 2 days. Cells are collected bycentrifugation at 3500×g for 10 minutes. The culture supernatants arediscarded and the cells are re-suspended in sterile deionized water tothe volumes of the original cultures. AIP0323, a mutant of AIP0069 whichdoes not have antifungal activity, is included as a negative control.

Ureidinospores of Phakopsora pachyrhizi are harvested by vacuum suctionfrom leaves infected with the fungus that manifest erupted pustules. Thespores are re-suspended in water at 10{circumflex over ( )}5/mL andinoculated onto detached soybean leaves as an aerosol using an airbrushusing techniques known in the art (Twizeyimana, M., and Hartman, G. L.2010. Culturing Phakopsora pachyrhizi on detached leaves andurediniospore survival at different temperatures and relativehumidities. Plant Disease 94:1453-1460).

Mixtures of AIP1620 cells re-suspended in water and fungicidal activeingredients are prepared in various ratios comprising 10{circumflex over( )}6, 10{circumflex over ( )}7, 10{circumflex over ( )}8, 10{circumflexover ( )}9 or 10{circumflex over ( )}10 AIP1620 cells/mL, mixed withfungicidal active ingredient at 1/10×, ⅓×, ½×, or 1× normal field userate, calculated by converting the field rate from the published labelto g/mL, based on an assumption about spray volume per hectare or acre.

The inoculated soybean leaves are treated the biocontrol agent at thetiters above, and with the fungicides at the rates above, as well aswith mixtures of biocontrol agent and fungicide in various combinationsat the titers and rates specified above. In addition, some inoculateddetached leaves are left untreated, or treated with AIP0323 as controls.At least 3 leaves (or leaf segments) are used for each treatment ofbiocontrol agent, chemical, or mixture. The detached leaves areincubated in high humidity in a growth chamber on a 12 light, 21° C./12hour dark, 23° C. After 10-14 days, the leaves are observed and scoredaccording to the number of visible uredinia/cm^2.

Colby's equation is used to determine the fungicidal effects expectedfrom the mixtures. (See Colby, S. R., Calculation of the synergistic andantagonistic response of herbicide combinations. Weeds 1967, 15, 20-22,herein incorporated by reference in its entirety.) The followingequation is used to calculate the expected activity of mixturescontaining two active ingredients, A and B:Expected=A+B−(A×B/100)

-   -   A=observed efficacy of active component A at the same        concentration as used in the mixture;    -   B=observed efficacy of active component B at the same        concentration as used in the mixture.        Representative synergistic interactions, including application        rates employed and resulting disease control are observed and        recorded as follows:

% DC=Percent disease control

% DC Obs=Percent disease control observed

% DC Exp=Percent disease control expected

Synergism factor=% DC Obs/% DC Exp

Example 19 Selection of a Population of the Biological Control StrainPseudomonas fluorescens AIP000069 that has Acquired Resistance to theHerbicide Glufosinate

50 microliters of AIP000069 culture, grown in 0.5× LB for 24 hours at28° C., was spread onto plates containing M63 Plus medium with 0 or 100mM Glufosinate. The M63 Plus medium consisted of 13.6 g KH₂PO₄, 9.92 gC₆H₁₂O₆, 2 g (NH₄)₂S0₄, 5.5 mg CaCl₂, 0.278 mg FeSO₄.7H₂O, and 10.16 mgMgCl₂.6H₂O per liter of deionized water. In the absence of Glufosinatenumerous bacterial colonies (a lawn) were visible after incubatingplates for 2 days at 28° C. In the presence of 100 mM Glufosinate nocolonies were visible after a similar incubation, however, afterextended incubation of several days a single colony grew. This colonywas streaked to isolation on an M63 agar plate containing 100 mMGlufosinate. The resulting isolate was named AIP050999. Growth ofAIP050999 was compared to the parent strain, AIP000069, and a glyphosateresistant version of the strain AIP001620. Results are summarized in thetable 15 below.

TABLE 15 Growth of strains on M63 Plus agar medium in the presence andabsence of glufosinate. Strain 0 mM Glufosinate 100 mM GlufosinateAIP000069 +++ − AIP001620 +++ − AIP050999 +++ +++

All publications and patent applications mentioned in the specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. A method of producing an improved biocontrolagent, said method comprising in vitro modifying a Pseudomonas bacteriumhaving biocontrol activity and susceptibility to a herbicide to beresistant to the herbicide, wherein the in vitro modifying comprisesculturing and selecting the Pseudomonas bacterium in the presence of theherbicide such that the improved biocontrol agent is created by thePseudomonas bacterium undergoing genetic mutation to develop resistanceto the herbicide and survives, thereby producing a culture of theimproved biocontrol agent, wherein the herbicide is selected from thegroup consisting of glyphosate, glufosinate (glutamine synthaseinhibitor), sulfonylurea, and imidazolinone herbicides (branched chainamino acid synthesis inhibitors).
 2. The method of claim 1, wherein saidPseudomonas bacterium is a Pseudomonas fluorescens bacterium or aPseudomonas chlororaphis bacterium.
 3. A modified biocontrol agentwherein said modified biocontrol agent has been selected in vitro underherbicide pressure and is resistant to said herbicide, wherein saidbiocontrol agent is a Pseudomonas bacterium having biocontrol activity,and wherein the in vitro selection under herbicide pressure comprisesculturing a parent biocontrol agent that is susceptible to the herbicidein the presence of the herbicide such that the modified biocontrol agentis created by the parent biocontrol agent undergoing genetic mutation todevelop resistance to the herbicide and survives to produce a culture ofthe modified biocontrol agent, wherein the herbicide is selected fromthe group consisting of glyphosate, glufosinate (glutamine synthaseinhibitor), sulfonylurea, and imidazolinone herbicides (branched chainamino acid synthesis inhibitors).
 4. The modified biocontrol agent ofclaim 3, wherein said Pseudomonas bacterium is a Pseudomonas fluorescensbacterium or a Pseudomonas chlororaphis bacterium.
 5. The method ofclaim 2, wherein said Pseudomonas bacterium comprises a strain depositedas NRRL No. B-50897.
 6. The modified biocontrol agent of claim 4,wherein said Pseudomonas bacterium comprises a strain deposited as NRRLNo. B-50897.
 7. A modified biocontrol agent comprising a Pseudomonasbacterium strain having biocontrol activity and resistance to glyphosateand deposited as NRRL No. B-50897, wherein a parent of said strain wasmodified in vitro to have said resistance to glyphosate, wherein the invitro modification comprises culturing and selecting the parent of saidstrain in the presence of glyphosate such that the modified biocontrolagent is created the parent undergoing genetic mutation to developresistance to glyphosate and survives to produce a culture of themodified biocontrol agent.